The Feeding Habits and Growth Rates of Some Fresh-Water Ciliates Found in Activated-Sludge

Plants
Author(s): C. R. Curds and J. M. Vandyke
Source: Journal of Applied Ecology, Vol. 3, No. 1 (May, 1966), pp. 127-137
Published by: British Ecological Society
Stable URL: http://www.jstor.org/stable/2401669
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THE FEEDING HABITS AND GROWTH RATES OF SOME
FRESH-WATER CILIATES FOUND IN ACTIVATED-SLUDGE
PLANTS
BY C. R. CURDS AND J. M. VANDYKE

WaterPollutionResearchLaboratory,Stevenage

Therehas been a greatdeal of speculationas to therole oftheciliatedprotozoa in the

purification of sewagebytheactivated-sludge process,butthesignificance or importance
of theseorganismshas not yetbeen clearlydefined.
Many observationshave indicatedthatwhenlargenumbersof protozoa are present
theclarityoftheeffluent is enhanced,and Watson(1945) and Curds(1963) havesuggested
the methodsby whichcertainciliatesinducethe flocculationof suspendedmatterand
bacteria.Bahr (1954) suggestedthat the predatoryactivityof protozoa was a major
factorin reducingto a verysmall residuethe populationof bacterialeftin theeffluent
aftertreatment of the sewageby the activated-sludge process.The predationof ciliates
upon bacteriahas been studiedfromtimeto timebut theciliatesso farinvestigated by
othersare not thosenormallyfoundassociatedwithactivated-sludge plants.Successions
ofbacteria(Allen 1944)and protozoa(Curds 1966)havebeen shownto occurduringthe
development of an activatedsludge,and Curds(1966) postulatedthat,ifciliateswereto
feedpreferentially upon different bacteria,theremightbe a linkbetweenthesuccessions
of thesetwo groupsof micro-organisms. Earlier,Hetherington (1933) had suggested
that the successionsof protozoa in laboratoryhay infusionswere due in part to the
presenceof theirindividualfood organismsin additionto changesin the chemistry of
the environment.
As a firststepin an attemptto elucidatethepredatory oftheciliatedprotozoa
activities
in activatedsludge,fiveciliates,separatedfromthisenvironment, wereculturedin the
laboratoryand presentedwitha range of bacteria,all of whichhad been reportedto
occur in sewage-treatment plants,as the food organisms.Using thesemethodsit was
hoped to discoverwhichbacteriatheprotozoain activatedsludgemightfeedon and at
a laterdate to estimatetherateof removalof bacteriafromsewageby thepredationof
ciliatedprotozoa.

MATERIALS AND METHODS

Sourceofprotozoa
Clonal culturesof thefivespeciesof ciliateused in thisinvestigation
originatedfrom
samplesand had been grownin laboratoryinfusionsfor2-3
various activated-sludge
years.

Protozoanstock-culture
media
A mediumof0 03 % (w/v)coagulatedeggyolkand 0.0300 (w/v)driedlettuceinfusion
was suitable for the growthof ParameciumcaudatumEhrenberg,Histriculusvorax
Corliss,and Epistylisplicatilis buta 005%0(w/v)infusionofGlaxo 'Complan'
Ehrenberg,
in distilledwaterproved a bettermediumfor the cultureof Parameciumcaudatum,
Histriculusvorax,Vorticellamicrostoma
Ehrenberg,and OperculariacoarctataClaparede
and Lachmann.
127

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128 ciliates
Feedinghabitsoffresh-water
Sourceof bacteria
The bacteria,whichhave all been reportedpresentin sewage,in activatedsludge,or
wereobtainedfromtheNationalCollectionofIndustrialBacteria,
in percolatingfilters,
TorryResearchStation,Aberdeen.Stock culturesof thebacteriaweregrownon slopes
of 'Oxoid' blood agar base No. 2, at 300 C.

Estimationof ciliatedivisionrateby theisolationculturetechnique

(i) Preparationof culturevessels.Five glass rings,2 cm diameterand 1 cm deep,were
sunkinto a 5 mmlayerof moltenagar (2 5 g agar, 0 05 g NaCl, 0 05 g Liebig's beef
extractin 100 ml distilledwater),containedin a 6 5 cm diameterPetridish.Dishes con-
tainingglass ringsand agar were sterilizedby autoclaving(15 min at 15 lb/in2),and

One
cea pinked Divisionfor ix cels picked
Ma d rout
inteo-and / 24 h at 250 C t, two Introduc
duced into ring into each of three
of bacteriars rings

FsG.u1.pMetiodusedfortesting
triplicateisolationculture. Divisionfor

division
rates
calculated
Celsscount ineach
ed ring,
t

Mean division rates of

J{w0y2t/t

y
Divisionftor Ti
\C(
250
p

p ix fv s c pt s e cc ouAtfarom
itked
each of three rings, two
introduced into each ofthree
freshriWngs
FIG. 1. Method used for testingtriplicateisolation culturetechnique.

subsequentlyallowed to cool on a horizontalsurface.This proceduredividesthe agar

plate intofivesterilecompartments, each of approximately 1 5 ml capacity.An appro-
priate bacterium was streaked on to the surfaceof the agar base enclosedwithinone ring
and flooded with 1P0 ml sterile
borehole water.
Althoughthismethodprovedto be ideal forthe cultureof all theprotozoanspecies
studied,therewas some difficulty in countingperitrichs in thesecompartments. It was
found easier to cultureperitrichson cavityslides so that the organisms could then be
counteddirectly usinga mechanicalstageunderthelow powerobjectiveofa microscope.
One drop of moltenagar was pipettedintothecavityof a clean slidewhichwas then
placed insidea moistchamberand autoclavedinsitu.Aftersterilization and cooling,the
appropriatebacteriawerestreakedon to theagar and thecavitywas floodedwithsterile
boreholewater.
The concentrations of bacteriaproducedby both of the above methodsprovedto be
in excessof the quantityrequiredby theciliates.
(ii) Theisolationculturetechnique. SinceMaupas (1888) theisolationculturetechnique

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 C. R. CURDS AND J. M. VANDYKE 129
has beenused by a largenumberofworkersas a methodof estimating thedivisionrates
of protozoa,but thisand subsequentmethodsdid not includeanymeansof replication.
In thepresentinvestigation theisolationculturetechniquewas modifiedin sucha wayas
to providethreereplicatedivisionratessimultaneously.
As a precautionarymeasure,the triplicateisolation culturetechniquewas tested
statistically
as follows(Fig. 1). A ciliatewas pickedoutfromthestockcultureand excess
bacteriawereremovedby a seriesof threewashesin sterileboreholewater.The washed
ciliatewas introducedinto a ringcontaininga cultureof a knownbacteriumand left
24 h at 250 C to divide.Six of theresultingdaughtercellswerepickedout and washed,
and twowereintroduced intoeach ofthreefreshly preparedringsofbacterialsuspension.
After24 h incubationat 250 C, six ciliatesfromeach of the culturesso obtainedwere
washed and introducedinto threeringsof freshlypreparedbacterialsuspension,two

\clae
/ne
cell
picked cells
\ /Division
\ /ix
for picked
on
sus
pensi
out and intro- 24 h at 25 C out two
introduced
duced intoring into each of three
of
bacteriar rings

Divisionfor
24h at 25tC

Cent s d
counnte Ce.lscounted
d

rson ( ined sthea of times Six cetspicker

ied< i.e
out fromonering 24 h at 250C J out fromoneri n
two introducedinto ~~two introducedinto
each ofthreetresh eachof threefresh
rings rings
FIG. 2. Triplicate isolation culture method employed for the estimationof the division
rate of ciliated protozoa feedingon excess of bacteria.

24 h incubation,thenumbersofciliatesin each ofthe

ciliatesto each ring.Aftera further
nineringswerecounteddirectly usinga low powerstereo-microscope.The dailydivision
rates,R, (definedas the averagenumberof timesthepopulationdividesper day, i.e. n
organisms becomen.2R in I day),ofthenineciliatecultures,
whichderived froma single
cell,werecalculatedusingtheformula:

R log B-log A
log 2
whereA = numberof ciliateson Day 1, and B = numberof ciliateson Day 2.
It maybe notedthat
R = l/tD

wheretD is the mean generationtimeof the organismin days.

The meandivisionratesofthethreesetsofthreeringcultureswerecalculatedand these
means, X, Y and Z, comparedwitheach otherby the t-test('Student' 1925). These
comparisons-i.e. X withY, X withZ and Y withZ-each showedthatthemeanswere
not significantly fromeach other,P>0 80 in each case. Use of the t-test
different
be normalas appearedto be the case.
requiresthatthe distribution

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130 ciliates
Feedinghabitsoffresh-water
The procedureused throughout thisinvestigationwas similarto thatdescribedabove
(Fig. 2). Because theresultsof thepreliminarytestshowedthatthemean divisionrates
of the threeringcultureswerenot significantly different, two of the culturesweredis-
continuedat thispoint,whilethethirdremaining linewas carriedon by transferringsix
of the cells to threefreshringsby the usual procedure.By thismethodthe standard
deviationof the mean dailydivisionrate of threereplicateciliateculturescould be cal-
culatedrepeatedly, overa numberof weeksifnecessary.

Preparationofknownconcentrations of bacterialsuspensions
Bacteria, from
harvested an agar slope, weresuspendedin sterileboreholewaterand
dilutedto therequiredconcentrationby matching suspensionswithcalibrated'Wellcome'
opacitytubes.Opacity tubeswere calibratedfor each the
bacterialspeciesby estimating
numberof viablebacteriain suspensions of known opacity.

Viablebacteriacounts
Countsoftotalviablebacteriawerecarriedout by serialten-folddilutionsin borehole
water,followedby platingon blood agar base No. 2 and incubationat 300 C, following
thetechniquedescribedby Miles & Misra (1938).

RESULTS

Effectsofnineteeni species)of bacteriauponfiveciliatespecies

strains(fifteen
Followingthetriplicateisolationcultureprocedurealreadyoutlined,nineteenstrains
of bacteriawere presentedindividuallyas the sole particulatefood source to the five
speciesof ciliatedprotozoa.Soluble organicmatterwhichdissolvedout of theagar base
intosterileboreholewaterdid not supportthegrowthof theprotozoain the absenceof
bacteria.Fromtheresultsof theseexperiments (Table 1) it willbe seenthatthebacteria
chosencan be dividedintofivecategories,bytheeffects thattheyhave upon theciliates.
(i) Verytoxic bacteria.Chrombacterium violaceumwas the only bacteriumof those
chosenwhichkilledciliateswithin3 h. C. violaceumcontainsthevioletpigment'violacein'
whichwas isolatedand purified by Strong(1944) and is knownto be toxic,ifingested,to
various protozoa (Burbanck1942; Kidder & Stuart 1939). If a 2-day cultureof this
bacteriumis membrane-filtered, the bacteria-freefiltrateis stillhighlytoxicto ciliates.
Further, microstoma
ifcystsof Vorticella are placedin a suitableculturemedium95 + 50
of the cystswillexcystwithin2 h, but none excystfollowinga 2 h periodof immersion
in a cultureof Chromobacterium violaceum.It would appear,as violaceinis not water-
soluble, that a second toxin is secretedinto the culturemediumby this bacterium.
RecentlyGroscop & Brent(1964) reportedthatcertainsmallfree-living amoebae were
killedby C. violaceumbeforeingestionof thebacteriumhad takenplace.
(ii) Toxicbacteria.In thiscontext,toxicbacteriaare definedas thosewhichkillciliates
within5-24 h ofcontact.The bacterium Proteusvulgaris was theprincipalexampleofthis
typeand provedto be toxicto threeciliates.P. vulgarisis non-pigmented and it is not
clearin thiscase ifone or moretoxinsare involved.Eithera solubletoxinis secretedby
the cell or the bacteriaalterthe culturemediumin some way whichmakesit lethalto
Vorticellacysts.Similarly, of membrane-filtered
filtrates culturesof Proteusvulgarisare
toxicto adult ciliates.
(iii) Slightlytoxicbacteria.The entericbacteriaEnterobacter cloacae and Escherichia
coli are toxicto Parameciumcaudatumwhenpresentedin highconcentrations. In lower

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 C. R. CURDS AND J. M. VANDYKE 131
concentrationsthesebacteriaare non-toxicbut do notsupportthe growthof the ciliate
cells for more than a few days. Johnson(1933) reportedthat a heavy inoculumof
5 -

7
b
S
5
4
O2 -
4
3-
2 -

6 2

5T (d)
30
4-
X4-r
3-
-0
3-0152
2-

3 - e
2-

0 5 1 52

Time (days)
witha singlebacterial
FIG. 3. Daily divisionratesoffivespeciesofciliatewhenpresented
speciesas thesolefoodorganism. areshown.
Mean divisionratesand standarddeviations
(a) Parameciumcaudatumfeedingon Klebsiella aerogenes,(b) Histriculusvorax feedingon
Pseudomonasovalis, (c) Vorticellamicrostomafeedingon Bacillus cereuts,(d) Opercularia
coarctatafeedingon Klebsiella aerogenes,and (e) Epistylisplicatilisfeedingon Alcaligenes
faecalis.

Pseudomonasfluorescenskilledthe hypotrichOxytrichawhilelower concentrations of

thebacteriumprovedto be suitableas a sourceof food.
(iv) Unifavourablebacteria.Many speciesofthechosenbacteria,whenpresentedto the
ciliatesas thesolefoodorganism, eveninhighconcentrations,
werefoundto be non-toxic,

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132 Feedinghabitsoffresh-water
ciliates

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 C. R. CURDS AND J. M. VANDYKE 133
but ciliategrowthwas supportedforless than 8 days. In manycases, ciliatesfedupon
unfavourablebacteriawere observedto become smallerand smaller,showingsignsof
starvation.The bacteriumAzotobacterchroococcuminduced monsterformationin
Parameciumcaudatum.Monsterstook theformof chainsof cellsformedby incomplete
transversefission,each of whichpossessedtwo contractilevacuoles and a mouth.It
would appear that unfavourablebacteria,althoughnon-toxic,do not fulfilthe total
dietaryrequirements of theciliates.
Emptyfood vacuoles wereoftenobservedinP. caudatumand Histriculus voraxwhen
were
theseciliates fed on unfavourablebacteriaforperiodsexceeding 3 days. Observation
of Parameciumcaudatum,using phase-contrastmicroscopy,showed that although
bacteriaweretakenintotheformingfood vacuole,immediately priorto its completion
the quadruluscilia reversedtheirbeat so thatparticleswere wafted out of the vacuole
and buccal cavityin a rejection current.This evidencesupports the views of others
(Losina-Losinsky1931; Grittner 1951; Bahr 1954) who believethatsome ciliatespossess

Table 2. Minimumdailydivisionrate,R, and maximumdoublingtime,tD, necessary

plant
forsurvivalofprotozoanspeciesin threetypesofactivated-sludge
Typeofplant
High-rate Extended
partialtreatment Conventional aeration
Sewageretentiontime(h) 3 12 72
Freeswimmers geotactic)
(negatively
R (divisions/day) 46-5 5-8 0-96
tD(days) 0 043 0-172 1-03
Attachedforms
R 0 37 0-17 001
tD 2-7 5 95 93-2
takeintoaccounttheflowratesofthesewage;in thecase of
Thesecalculations
theattachedforms,theretention timesof themixedliquor; in the case of the
free-swimming forms,theretentiontimesof the sewage,the sludgegrowth,the
proportionof sludge(and henceprotozoa)returnedto theaerationtankand the
proportionofprotozoalostwhenthesludgeis led offfordisposal.

a meansof selection.The formation of emptyfoodvacuolesbyP. caudatumhas another

functionwhichwillbe discussedlater.
(v) Favourablebacteria.These bacteriaare definedas thosewhichsupportthegrowth
and divisionofciliatedprotozoa,as thesolefoodorganism, fora periodofatleast3 weeks.
It willbe seenfromTable 1 thattheeffect thatone bacterialspecieshas on one ciliate
can be quite differentfromits effecton anotherspeciesof ciliate.Escherichiacoli, for
example,is slightlytoxic to Parameciumcaudatum,unfavourableto Vorticellamicro-
stomaand Opercularia coarctata,yetfavourableto Epistylisplicatilis vorax.
and Histriculus
Divisionratesofprotozoafeedingonfavourablebacteria
feedingon excessoftheirfavourablebacteria
The dailydivisionratesofthefiveciliates-
wereestimatedusingthetriplicateisolationculturetechnique.Significant changesin the
divisionratewerenotedfromdayto day in all speciesofciliate(Fig. 3). Changesin daily
divisionrate have been noted by a numberof authorsincludingWoodruff& Baitsell
(191la, b) and Sonneborn(1954), and the declinein divisionrate has been correlated
withautogamy,a meioticre-organization processof the micronucleus.Because of such
themeansoffifteen
changesin division-rate dailydivisionrateswereestimated
triplicate
and standarddeviationscalculated,values forthesebeinggivenin Table 1.

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134 ciliates
Feedinghabitsoffresh-water
ofthedifferences
The significance ofthemeandivisionratesoftheciliatesfeedingupon
theirfavourablebacteriahavebeentestedusingthet-test('Student'1925).Ifa probability
of less than002 is acceptedas illustrating
a significant
difference betweenthemeansof
the divisionrates,thenin a numberof cases the 15-daymean daily divisionrate of a
whenfed on different
ciliatevariessignificantly bacteria.This is in agreementwiththe
workof a numberof previousauthors(see Burbanck1942).

Effectof differentbacterialconcentrationson thedivisionrateof Parameciumcaudatum

Known concentrations of the favourablebacteria Klebsiellaaerogenesand Bacillus
cereuswerefedto Parameciumcaudatum.No significant differencein the daily division
ratewas notedin cellsfeedingon bacterialconcentrations lyingwithintherange20-800
whichis the approximaterangeof concentrations
millionviable bacteriaper millilitre,
used in theisolationculturetechnique,butwhenP. caudatumand Histriculus voraxwere
fed on bacterialsuspensionsabove a concentration of 200 millionviable bacteriaper
intermittent
millilitre, phasesofemptyand normalfoodvacuoleformation wereobserved.
Concentrations above 800 millionviable bacteriaper millilitre
caused anaerobiccondi-
tionswithinthe suspensionand subsequentdeathof theciliates.
Settling tank

Sewage E ffuent

- Aeration tank
containing mrixed liquuor

Sludge disposal

Returned sludge
FIG.4. Flow diagramof a typicalactivated-sludge
plant

DISCUSSION
In theactivated-sludge process(Fig. 4), as sewageentersthesystem,effluent leavesat an
equal rate,carryingwithit those protozoa swimmingfreelyin the liquor,whilethose
protozoa closelyassociatedwithor attachedto the sludgeflocculeswill be lost when
excesssludgeis led offfordryingand disposal.Therefore, in orderto survivewithinan
activated-sludge plant,theprotozoamustdivideat a ratehighenoughto withstand these
continuallosses. Protozoa enteractivated-sludge plantsvia the sewage and these will
survive,die or be 'washedout' accordingto theenvironmental conditionsin the aeration
tanks.
In conventional plants,theliquoris retainedin thesystemforapproximately 12 h, and
ifitis assumedthatall free-swimming protozoamigrateto thesurfacein thefinalsettling
tanks,theseorganismsmustdivideon averageat least 5-8 timesper day if theyare to
survive(Table 2). On the otherhand if the organismsbecome uniformly distributed
betweenreturnedsludgeand effluent the maximumnumberof divisionsper day need
onlybe 2-9. It is knownthatParameciumcaudatumis negatively geotactic(Curds 1963)
and willtherefore tendto swimto thesurface,butitmaywellbe thatotherfree-swimming
protozoaare eitherpositively geotacticor haveno tacticreactionstowardsgravity. High-
rateplantshavingshorterliquorretention times,willtendto selectthosefree-swimming
ciliateswhichcan divideat correspondingly higherrates.Sludge is retainedforlonger
periodsoftimethanliquorand so thoseprotozoaattachedto thesludgesurfacecan have

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 C. R. CURDS AND J. M. VANDYKE 135
slowerdivisionratesthan the free-swimmers and yet be maintainedwithinthe plant.
Table 2 showsexamplesof the calculatedminimumnumbersof divisionsper day, and
corresponding maximumdoublingtimes,of whollyfree-swimming protozoa and of
attachedformsin threetypes of activated-sludge plant under operatingconditions
appropriateto thetreatment of domesticsewage.
Comparingthedata of Table 1 withthoseof Table 2 it is evidentthat if the division
ratesoftheciliatesare similarin activatedsludgeto thosefoundin experimental cultures
thenall threeperitrichs, Vorticella, Operculariaand Epistylis(all attachedforms),and the
hypotrich, Histriculus, whichcrawlson the surfaceof the sludge,would be expectedto
survivein any of theplantsoperatingundertheconditionsgivenin Table 2. In practice
however,Operculariaspp. are the onlyciliateswhichhave been observedin high-rate
plants.Because of its relativelylow growthrate,Parameciumcaudatumshould survive
onlyinplantsoperatingat low ratesofflow,and observations overa numberofyearshave
substantiated thishypothesis.
The bacterialpopulationin activatedsludgewillbe determined by a numberof inter-
actingfactorsincludingthe growthrate of the organisms,the natureof the inflowing
sewage,and theoperatingconditionsin theplant.Downing,Painter& Knowles(1964),
forexample,havedemonstrated thewaysin whichsomeofthesefactorsaffect theecology
of the nitrifying bacteriumNitrosomonas in activatedsludge.In the same way the pro-
tozoan populationwillbe determined eitherdirectlyby thephysicaland chemical con-
ditionsor indirectly by the populationdensitiesof the bacterial flora upon which they
feed; onlythoseprotozoa whichpreyupon the typesof bacteriathatcan developwill
flourish.
Daily changesin the divisionrate of a ciliateline have been reportedpreviouslyby
a numberof authors,butthesereportsare opento criticism sinceno attemptwas madeto
examinethe statisticalsignificance of the results. Results obtainedusingthe triplicate
isolationculturetechnique, however, have verified the occurrence of significant daily
changesin the divisionrate of ciliates. There is considerable evidence, reviewed by
Sonneborn(1954), suggesting that the period of division rate decline and autogamy are
closelyrelated.
If such changesin divisionrate occurwithinnaturalpopulations,theireffectis likely
to be greaterin an environment whichoperatesbythecontinuous-culture system.A high
divisionrate could mean the appearance and accumulation of a ciliate speciesin the
of
protozoanpopulation activatedsludge, whereas a lowering of the division ratecould
in
result the 'washout' of a species. In practice, the ciliate population of an activated
is
sludge often observed to change completely over short periods of time for no apparent
reason; but clearly this might be the result of a change in the division rates of the ciliate
speciesmakingup that population.
Cutler & Crump (1924) reportedthat the divisionrate of Colpidiumcolpoda was
dependenton theconcentration of bacteriaavailable,but Harding(1937) statedthat in
Glaucomafissionratewas not influenced by bacterialconcentration in therangeof06-7
millionbacteria/mm3. The results of Cutler & Crump (1924) also illustrate thispoint; if
growth rateis plotted against concentration of available bacteria, division rate gradually
becomesindependent whenfood supplyis presentin highconcentrations. In thepresent
work,bacterialfood supplywas alwayskeptin excessso thatit is not surprising thatno
significant change in the fission rate of a ciliate fed upon different concentrations of
bacteriawas observed.
The formation of emptyfood vacuolesbyParameciumcaudatumand Histriculus vorax

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136 Feedinghabitsoffresh-water
ciliates
would appearto have two functions. The firstis as a mechanismfortherejectionof un-
suitablebacteria,a mechanismwhichcomes into operationonlyaftersome days. This
supportstheviewheld by Grittner (1951), who regardedemptyfood vacuole formation
in Parameciumas a 'negativereactiontowardsuselessor harmfulsubstances'.However,
furtherobservationsdescribedin the presentpaper have indicatedthat emptyfood
vacuolesformin thepresenceof highconcentrations offavourablebacteria,and thereis
an explanationforthis.Bozler(1924) observedthatin 'completely dust-free
water'ciliates
did notformfoodvacuolesat all, butlaterMast (1947) disagreedwiththisstatement and
reportedthatalthoughfoodvacuolesare formedin theabsenceofparticulatematter, the
numberof vacuoles formedis relatedto the numberand qualityof particlesin the
medium.Seaman (1961) agreedwiththe latterview and remarkedthat in the case of
Tetrahymena thenumberoffoodvacuolesproducedin theabsenceofparticulatematter
is fartoo fewto supplythenutritional requirements of theorganism.These reportssug-
gestthatalthougha certainminimumnumberof vacuolesis formedin particulate-free
media,food vacuoleformation is a functionoftheconcentration offavourableparticles
present.Thereforein high concentrations of favourablebacteriaa strongstimulusis
exertedupon theciliateto formmanyfoodvacuoles,butbecausegrowthrateis indepen-
dent of food supplyat theseconcentrations, theremustbe a point beyondwhichthe
ciliatecannotcope withthe bacterialintake.Intermittent formationof fulland empty
foodvacuoleswouldtherefore providea regulatory mechanism forthecontrolofingestion.
It is suggestedtherefore
thatifthebacterialfoodsupplyis in highenoughconcentrations
thensome ciliatedprotozoa cease to be continualfeedersand controltheirparticulate
food intakeby an alternating cycleof emptyand fullfood vacuoles.

ACKNOWLEDGMENTS
The authorsare indebtedto Mr G. Knowlesforadvice on the minimumdivisionrates
necessaryto retainprotozoain activated-sludge
plants.This studywas supportedbythe
Ministryof Technologywhilethe seniorauthorheld a ResearchFellowship.

SUMMARY
1. Five fresh-waterciliatedprotozoahave been culturedmonoxenically upon nineteen
strains(fifteenspecies)of bacteriawhichinhabitsewage-treatment plants.
2. The bacteriahad different effects
upon the chosenciliates;some weretoxic,some
werenon-toxicbutwould not supportgrowth,and some supportedciliategrowthforat
least 3 weeks.
3. The fiveciliatesoftenreacteddifferently to the same speciesof bacteria.
4. The divisionrates of the ciliatesfeedingupon highconcentrations of favourable
bacteriawerecalculatedusinga triplicateisolationculturetechnique.The divisionrate
of a ciliateunderthese conditionsvaries significantly fromday to day. The mean of
fifteendaily divisionrates of a ciliate speciescan varysignificantly when it is fed on
different speciesof bacteria.
5. The formationof emptyfood vacuoleshas been observedin Parameciumcaudatum
and Histriculusvorax.Emptyvacuole formation,it is suggested,is both a methodof
rejectingunfavourablebacteriaand a mechanismfor limitingthe rate of ingestionof
favourablebacteriawhentheseare presentin excess of the requirement formaximum
growthrate.

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 C. R. CURDS AND J. M. VANDYKE 137
6. The minimum dailydivisionratesnecessaryforthesurvivalofprotozoain activated-
sludgeplantsoperatingat threeflowrateshavebeencalculatedand havebeencompared
withthedivisionratesoftheciliatesmeasuredusingculturetechniques.This comparison
has demonstratedthat while Parameciumcaudatumcould be expectedto survivein
activated-sludgeplantsoperatingat low flowrates,the otherciliatesinvestigated could
be expectedto survivein all threeplants.In practiceOperculariaspp. are theonlyciliated
protozoawhichsurvivein plantsoperatingat highflowrates.

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(Received3 June1965; revisionreceived2 August1965)

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